Volume 29, Issue 2, Pages 263-270 (February 2008) Conserved GU-Rich Elements Mediate mRNA Decay by Binding to CUG-Binding Protein 1  Irina A. Vlasova, Nuzha M. Tahoe, Danhua Fan, Ola Larsson, Bernd Rattenbacher, Julius R. SternJohn, Jayprakash Vasdewani, George Karypis, Cavan S. Reilly, Peter B. Bitterman, Paul R. Bohjanen  Molecular Cell  Volume 29, Issue 2, Pages 263-270 (February 2008) DOI: 10.1016/j.molcel.2007.11.024 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 GREs Are Functional Mediators of mRNA Decay (A) The shown GRE-containing sequences from the 3′UTR of c-jun, jun B, and TNFRSF1B were cloned into the 3′UTR of the pTetBBB β-globin reporter construct. The boxed sequences indicate the sequences of the ribo-oligonucleotides used for the binding reactions shown in Figure 2. The arrows indicate the single-nucleotide mutations (G to C) that were introduced to create the mutated GRE sequences. (B) HeLa Tet-off cells were transfected with the pTracer GFP expression construct and the pTetBBB β-globin reporter construct (No Insert) or with reporter constructs in which GRE-containing sequences (c-jun, jun B, and TNFRSF1B) or mutated sequences (mc-jun, mjun B, and mTNFRSF1B) shown in (A) were inserted into the 3′UTR. Doxycycline was added to the medium to stop transcription from the tet-responsive promoter, and total cellular RNA was collected at 0, 2, 4, and 6 hr time points. Northern blot analyses were performed to monitor GFP and β-globin (BG) mRNA levels. (C) The experiment shown in (B) was performed three times, and the northern blots were quantified using a phosphorimager. For each point, the intensity of the β-globin reporter band was normalized to the intensity of the GFP band, and the band intensity at the 0 hr time point was set at 100%. The percent of mRNA remaining was plotted over time. The error bars indicate the standard error of the mean (SEM) from the three experiments. Molecular Cell 2008 29, 263-270DOI: (10.1016/j.molcel.2007.11.024) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 CUGBP1 Binds Specifically to GRE Sequences with High Affinity (A) Cytoplasmic extracts from primary human T cells were mixed with a 32P-end-labeled ribo-oligonucleotide probe that contained a GRE sequence from the 3′UTR of the c-jun transcript in the absence (No Competitor) or presence of a 100-fold molar excess of the indicated unlabeled competitor ribo-oligonucleotides. The sequences of the GRE-containing ribo-oligonucleotides and mutated oligonucleotides are indicated as the boxed sequences in Figure 1A. Bands were visualized using a phosphorimager, and the position of migration of the predominant RNA-protein binding complex is indicated with an arrow. (B) RNA-protein gel shift assays were performed by mixing cytoplasmic extracts from primary human T cells with radiolabeled ribo-oligonucleotide probes that contained GRE sequences from the 3′UTR of c-jun, jun B, and TNFRSF1B or mutated GRE sequences (mc-jun, mjun B, and mTNFRSF1B) in the absence of antibody (No Antibody) or the presence of anti-CUGBP1 or anti-actin antibodies. The binding reactions were then separated by electrophoresis on 10% polyacrylamide gels under nondenaturing conditions. For each panel, the position of the supershifted band is indicated with an arrow. (C) Cytoplasmic extracts from primary human T cells (10 μg of protein) were incubated with 12 fmol of a 32P end-labeled c-jun GRE probe in the absence of unlabeled RNA or the presence of increasing amounts of unlabeled c-jun GRE RNA (12–2400 fmol in 2-fold increments). The binding reactions were then separated by electrophoresis on a 10% polyacrylamide gel under nondenaturing conditions. The lane marked “P” was loaded with probe alone. The position of migration of the probe bound to CUGBP1 is indicated with an arrow. (D) The experiment shown in (C) was performed three times, and the amount of bound c-jun GRE probe was quantified with a phosphorimager. The percent of maximal bound radiolabeled RNA was plotted against the concentration of total RNA in each reaction. Each point represents the mean and SEM from the three experiments. (E) HeLa Tet-off cells were transfected with the pTetBBB β-globin reporter construct (No Insert) or with reporter constructs in which the TNFRSF1B GRE sequence (TNFRSF1B) or the mutated sequence (mTNFRSF1B) shown in Figure 1A was inserted into the 3′UTR. Lysates from these transfected cells were incubated with protein G Sepharose beads that were precoated with anti-HA, anti-CUGBP1, or anti-PABP antibodies. RNA isolated from the input (I) and from the immunoprecipitation pellet (P) was assayed for the presence of β-globin (BG) or GAPDH transcripts using RT-PCR. As a control to detect possible contamination, lane 19 (H2O) contained all components of the RT-PCR reaction except for an RNA sample. Molecular Cell 2008 29, 263-270DOI: (10.1016/j.molcel.2007.11.024) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 siRNA-Mediated Knockdown of CUGBP1 Induced Stabilization of GRE-Containing Transcripts In (A) and (B), HeLa Tet-off cells were treated in two rounds with two independent siRNAs directed against CUGBP1 (siCUGBP1-A and siCGBP1-B) or a control siRNA directed against red fluorescence protein (siControl). In (C) and (D), HeLa Tet-off cells were treated in three rounds with smart pool siRNAs directed against CUGBP1 (siCUGBP1) or nontargeting siRNA (siControl). These cells were then transfected with the pTracer GFP expression construct and a pTetBBB β-globin reporter construct in which GRE-containing sequences (TNFRSF1B or jun B) were inserted into the 3′UTR. Doxycycline was added to the medium to stop transcription from the tet-responsive promoter, and total cellular RNA was collected at 0, 2, 4, and 6 hr time points. Northern blots were performed to monitor GFP, and β-globin (BG) mRNA levels. The experiments in (A) and (C) were performed three times, the northern blots were quantified using a phosphorimager, and the graphed results are shown in (B) and (D), respectively. For each point, the intensity of the β-globin reporter band was normalized to the intensity of the GFP band, and the band intensity at the 0 hr time point was set at 100%. The percent of mRNA remaining following the addition of doxycycline was plotted over time. The error bars indicate the standard error of the mean from the three experiments. (E) Lysates from HeLa cells were incubated with protein G Sepharose beads that were precoated with anti-HA or anti-CUGBP1 antibodies. RNA isolated from the input (I) and from the immunoprecipitation pellet (P) was assayed for the presence of c-jun, jun B, or GAPDH transcripts using RT-PCR. As a control to detect possible contamination, lane 5 (H2O) contained all components of the RT-PCR reaction except for an RNA sample. (F) HeLa cells were treated in two rounds with pooled siRNAs directed against CUGBP1 (siCUGBP1) or nontargeting siRNA (siControl). Actinomycin D was added to stop transcription and total cellular RNA was harvested at 0, 1, and 2 hr time points. The mRNA levels of c-jun and jun B were measured by real-time RT-PCR using transcript-specific primers, and transcript levels were normalized to the level of the HPRT transcript. The normalized level of each transcript was set at 100% at time zero, and the other time points were graphed relative to that value. Each point represents the mean and SEM from three independent experiments. Molecular Cell 2008 29, 263-270DOI: (10.1016/j.molcel.2007.11.024) Copyright © 2008 Elsevier Inc. Terms and Conditions